Encapsulated cells for hormone replacement therapy

ABSTRACT

A composition comprising microcapsules, the microcapsules containing both live mammalian ovarian granulosa cells and live mammalian ovarian theca cells, is described. In some embodiments, the granulosa cells and the theca cells are contained in separate microcapsules in the composition; in some embodiments, the granulosa cells and the theca cells are contained together in the same microcapsules in the composition The composition is can be used for estrogen, and optionally also progesterone, delivery, and hence is preferably free or essentially free of oocytes. Methods of using the same and pharmaceutical formulations containing the same are also described.

RELATED APPLICATIONS

This application is a divisional of and claims priority to U.S. patentapplication Ser. No. 14/002,504, filed Jan. 15, 2014, now U.S. Pat. No.9,283,251, which is a 35 U.S.C. §371 national phase entry of PCTApplication PCT/US2012/025892, filed Feb. 21, 2012, and published inEnglish on Sep. 13, 2012, as International Publication No. WO2012/121874, and which claims the benefit under 35 U.S.C. §119(e) ofU.S. Provisional Patent Application Ser. No. 61/449,267, filed Mar. 4,2011, the disclosure of each of which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention concerns compositions and methods for carrying outhormone replacement therapy in subjects in need of such treatment.

BACKGROUND OF THE INVENTION

Estrogens are versatile hormones, which are essential for variousphysiological functions in women. Reduced estrogen production from theovaries due to surgical resection, ablative therapy, or menopause leadsto various physiological consequences in women. Although hormonereplacement therapy is able to compensate for the loss of estrogenproduction, delivery through pharmacological means results inconsistently high serum concentrations. Clinical complications includeincreased incidence of heart disease and cancer. Accordingly, there is aneed for new methods and techniques for delivering estrogen.

SUMMARY OF THE INVENTION

A first aspect of the invention is a composition or pharmaceuticalcomposition comprising, consisting of, or consisting essentially ofmicrocapsules, the microcapsules containing both live mammalian ovariangranulosa cells and live mammalian ovarian theca cells. In someembodiments, the granulosa cells and the theca cells are contained inseparate microcapsules in the composition; in some embodiments, thegranulosa cells and the theca cells are contained together in the samemicrocapsules in the composition (e.g., in mixture with one another inthe same layer, core, or segment of the microcapsule). The compositionis intended primarily for estrogen, and optionally also progesterone,delivery, and hence is preferably free or essentially free of oocytes.

In some embodiments, the microcapsules comprise a core and an auxiliarylayer surrounding the core, with the core containing the granulosa cellsand the auxiliary layer containing the theca cells. In some embodiments,the microcapsules further comprising a first semipermeable layer betweenthe core and the auxiliary layer. In some embodiments, the microcapsulesfurther comprising a second semipermeable layer surrounding theauxiliary layer. In some embodiments, the microcapsules furthercomprising an external polysaccharide layer surrounding the secondsemipermeable layer. In some embodiments, the semipermeable layers areformed of a polycation (e.g., a polyamine).

In some embodiments, the microcapsules comprise a hydrogel such as apolysaccharide hydrogel (e.g., wherein the core comprises a hydrogelsuch as a polysaccharide hydrogel, and the surrounding layer comprises ahydrogel such as a polysaccharide hydrogel).

A further aspect of the invention is a method of administering estrogen,and optionally also progesterone, to a subject in need thereof,comprising administering the subject a composition as described hereinin a treatment-effective amount.

A further aspect of the invention is the use of a composition asdescribed herein for administering estrogen, and optionally alsoprogesterone, to a subject in need thereof, or for the preparation of amedicament for administering estrogen, and optionally also progesterone,to a subject in need thereof.

The present invention is explained in greater detail in the drawingsherein and the specification set forth below. The disclosures of allUnited States patent references cited herein are to be incorporated byreference herein in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: (Panel A) Schematic representation of encapsulation of ovarianendocrine cells. (Panels B-D) Flow cytometric analysis of purity ofisolated granulosa (Panel B) and theca cells (Panel C) purified using adiscontinuous percoll gradient (Panel D).

FIG. 2: Immuno-fluorescent staining for FSH-R & CYP19 (aromatase) ingranulosa cells (top panel) and for LH-R & CYP17A1 (17, 20 lyase) intheca cells (bottom panel).

FIG. 3: Phase-contrast microscopy and Scanning electron microscopyimages of encapsulated cells in alginate hydrogel microcapsules, showinghigh packing density of cells (Panels A and C) and low packing densityof cells (Panels B and D). This demonstrates the ability to achieve arange of packing densities of cells.

FIG. 4: Live/Dead staining of cells in the micro-capsules; Greenrepresents live cells and red represents the dead cells.

FIG. 5: (Panel A) Granulosa-microcapsules and Theca-microcapsules werecultured separately or co-cultured to see the effect on E₂ production.(Panel B) Effect of LH+FSH on E₂ production in co-culture system. (PanelC) Sustained E₂ production in long-term culture of granulosa cellmicrocapsule and theca cell microcapsule. *—denotes significance atP<0.05 compared to basal condition.

FIG. 6: Schematic diagram of a multi-layer microcapsule.

FIGS. 7A and 7B: Sustained E2 and progesterone production byTissue-construct in vivo. Each data point represents mean+SEM of 6values.

FIGS. 8A and 8B: (FIG. 8A) 17β-estradiol production by co-culturedgranulosa cells with theca cells in response to FSH and LH in 2D system.(FIG. 8B) Progesterone production by co-cultured granulosa cells withtheca response to FSH and LH in 2D system. Each data point representsmean+SEM of 6 values.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

“Subjects” as used herein are, in general, mammalian subjects. Whilehuman subjects are preferred, the subjects may in some embodiments beother animals, such as dogs and cats for veterinary purposes. Subjectsare generally female. While the subjects may be of any suitable age, thesubjects are typically adults and in some embodiments are menopausalfemale subjects.

“Treat” as used herein refers to any type of treatment that imparts abenefit to a subject, including but not limited to delaying the onset orreducing the severity of at least one symptom in the subject

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject to achieve thetreatments described herein, without unduly deleterious side effects inlight of the severity of the disease and necessity of the treatment.

1. Cells.

Cells used to carry out the present invention are, in general, livemammalian cells collected from a suitable donor. Donors are, in general,mammalian (e.g., human, dog, cat, rabbit, rat, mouse, monkey,chimpanzee, horse, pig, goat, sheep). The donor may be of the samespecies as the subject being treated, or of a different species. In someembodiments the donor may be the same subject undergoing treatment,where suitable cells were harvested from the subject and stored forsubsequent use.

Cells are isolated from donors and cultured for microcapsule productionas desired in accordance with techniques known in the art. See, e.g.,Sanjay K. Agarwal et al., Leptin Antagonizes the Insulin-Like GrowthFactor-I Augmentation of Steroidogenesis in Granulosa and Theca Cells ofthe Human Ovary, J. Clin Endocrinol Metab 84: 1072-1076 (1999); Jon C.Havelock et al., Ovarian granulosa cell lines, Molecular and CellularEndocrinology 228, 67-78 (2004); Jessica K. Wickenheisser et al., Humanovarian theca cells in culture, Trends in Endocrinology & Metabolism 17,65-71 (2006). In general, fresh tissue is divided by mincing, teasing,comminution and/or collagenase digestion. The desired cells are thenisolated from contaminating cells and materials by washing, filtering,centrifuging or picking procedures, and optionally cultured and/orcryopreserved as desired prior to encapsulation.

2. Microcapsule Production.

Encapsulation of live cells can be carried out in accordance with knowntechniques or variations thereof that will be apparent to those skilledin the art. See, e.g., U.S. Pat. Nos. 6,783,964 and 6,365,385 to Opara,the disclosures of which are incorporated by reference herein in theirentirety.

Microcapsules useful in the present invention optionally, but in someembodiments preferably, have at least one semipermeable membranesurrounding a cell-containing interior. The semipermeable membranepermits the diffusion of nutrients, biologically active molecules andother selected products through the surface membrane and into themicrocapsule core. The surface membrane contains pores of a size thatdetermines the molecular weight cut-off of the membrane. The membranepore size is chosen to allow the passage of estrogen, and in someembodiments progesterone, from within the capsule to the externalenvironment, but to exclude the entry of host immune response factors(where the encapsulated cells are not autologous). Such a semipermeablemembrane is typically formed from a polycation such as a polyamine(e.g., polylysine and/or polyornithine), as discussed further below.

In one non-limiting example embodiment of an encapsulation technique,U.S. Pat. No. 4,391,909 to Lim et al describes a method in which cellsare suspended in sodium alginate in saline, and droplets containingcells are produced. Droplets of cell-containing alginate flow intocalcium chloride in saline. The negatively charged alginate dropletsbind calcium and form a calcium alginate gel. The microcapsules arewashed in saline and incubated with poly-L-lysine or poly-L-ornithine(or combinations thereof); the positively charged poly-1-lysine and/orpoly-L-ornithine displaces calcium ions and binds (ionic) negativelycharged alginate, producing an outer poly-electrolyte semipermeablemembrane. An exterior coating of sodium alginate may be added by washingthe microcapsules with a solution of sodium alginate, which ionicallybonds to the poly-L-lysine and/or poly-L-ornithine layer (this serves toreduce any inflammatory response that may be provoked in the subject bycontact of the polycationic membrane to tissue). This technique produceswhat has been termed a “single-wall” microcapsule. A “double-wall”microcapsule can be produced by following the same procedure as forsingle-wall microcapsules, but prior to any incubation with sodiumcitrate, the microcapsules are again incubated with poly-1-lysine andsodium alginate.

In additional non-limiting examples of encapsulation methods, Chang etal., U.S. Pat. No. 5,084,350 discloses microcapsules enclosed in alarger matrix, where the microcapsules are liquefied once themicrocapsules are within the larger matrix. Tsang et al., U.S. Pat. No.4,663,286 discloses encapsulation using an alginate polymer, where thegel layer is cross-linked with a polycationic polymer such aspolylysine, and a second layer formed using a second polycationicpolymer (such as polyornithine); the second layer can then be coated byalginate. U.S. Pat. No. 5,762,959 to Soon-Shiong et al. discloses amicrocapsule having a solid (non-chelated) alginate gel core of adefined ratio of calcium/barium alginates, with polymer material in thecore. U.S. Pat. Nos. 5,801,033 and 5,573,934 to Hubbell et al. describealginate/polylysine microspheres having a final polymeric coating (e.g.,polyethylene glycol (PEG)); Sawhney et al., Biomaterials 13:863 (1991)describe alginate/polylysine microcapsules incorporating a graftcopolymer of poly-1-lysine and polyethylene oxide on the microcapsulesurface, to improve biocompatibility; U.S. Pat. No. 5,380,536 describesmicrocapsules with an outermost layer of water soluble non-ionicpolymers such as polyethylene(oxide). U.S. Pat. No. 5,227,298 to Weberet al. describes a method for providing a second alginate gel coating tocells already coated with polylysine alginate; both alginate coatingsare stabilized with polylysine. U.S. Pat. No. 5,578,314 to Weber et al.provides a method for microencapsulation using multiple coatings ofpurified alginate. U.S. Pat. No. 5,693,514 to Dorian et al. reports theuse of a non-fibrogenic alginate, where the outer surface of thealginate coating is reacted with alkaline earth metal cations comprisingcalcium ions and/or magnesium ions, to form an alkaline earth metalalginate coating. The outer surface of the alginate coating is notreacted with polylysine. U.S. Pat. No. 5,846,530 to Soon-Shiongdescribes microcapsules containing cells that have been individuallycoated with polymerizable alginate, or polymerizable polycations such aspolylysine, prior to encapsulation.

When desired, the alginate-polylysine microcapsules can be incubated insodium citrate to solubilize any calcium alginate that has not reactedwith poly-1-lysine, i.e., to solubilize the internal core of sodiumalginate containing the cells, thus producing a microcapsule with aliquefied cell-containing core portion. See Lim and Sun, Science 210:908(1980). Such microcapsules are referred to herein as having “chelated”,“hollow” or “liquid” cores.

When desired, the microcapsules may be treated or incubated with aphysiologically acceptable salt such as sodium sulfate or like agents,in order to increase the durability of the microcapsule, while retainingor not unduly damaging the physiological responsiveness of the cellscontained in the microcapsules. See, e.g., U.S. Pat. No. 6,783,964 toOpara.

One currently preferred method for the production of microcapsules isdescribed in O. Khanna et al., Synthesis of multilayered alginatemicrocapsules for the sustained release of fibroblast growth factor-1 J.Biomed. Mater. Res. Part A: 95A: 632-640 (2010).

Microcapsules may be of any suitable size, such as from 10, 20 or 30microns in diameter, up to 1000, 2000, or 5000 microns in diameter.Microcapsules may contain any suitable amount of cell. For example, insome embodiments, the granulosa cells are included in the microcapsulesin an amount of from 1,000 or 2,000 cells per microcapsule up to 1×10⁶,1×10⁸, or 1×10⁹ cells per microcapsule; and the theca cells are includedin the microcapsules an amount of from 1,000 or 2,000 cells permicrocapsule up to 1×10⁶, 1×10⁸, or 1×10⁹ cells per microcapsule.

Microcapsules of the present invention may be administered afterproduction, refrigerated and/or cryopreserved for subsequent use, and/orcultured for subsequent use, as desired. Microcapsules of the inventionmay be washed (e.g., in sterile physiological saline solution) prior toformulation and/or administration, as needed depending upon their mannerof production.

3. Formulation and Administration.

Microcapsules of the present invention may be administered per se orformulated for administration by any suitable technique, such as bymixing with sterile physiological saline solution. Microcapsules of thepresent invention may be administered to subjects as a treatment for anycondition in which estrogen replacement therapy is used. Themicrocapsules may be administered by any suitable technique, includingbut not limited to surgical implantation or injection (either of whichmay be carried out subcutaneously, intraperitoneally, intramuscularly,or into any other suitable compartment. Dosage of cells administered canbe determined in accordance with known techniques or variations thereofthat will be apparent to those skilled in the art. For comparison, inthe treatment of diabetes, the International Islet Transplant Registryhas recommended transplants of at least 6,000 cells per kilogram ofrecipient body weight, to achieve euglycemia. In the present invention,the number of cells implanted will depend upon the age and condition ofthe subject, the particular disorder being treated, etc. In someembodiments of the present invention, from 1,000, 2,000 or 3,000 cellsper kilogram of recipient body weight, up to 20,000, 40,000 or 60,000cells per kilogram recipient body weight, are administered.

Subjects or patients to be treated by the methods of the presentinvention include subjects afflicted with, or at increased risk of, oneor more of osteoporosis, hot flashes, irregular period, vaginal atrophy,vaginal and/or bladder infection, incontinence (e.g., urge incontinence,stress incontinence), fatigue, sleep disturbances, irritability, moodswings, depression, loss of muscle mass, increased fat tissue, thinningand loss of skin elasticity, loss of bone tissue, impaired cognitionetc., which may be associated with menopause, hysterectomy, ovarectomy,or other condition for which estrogen or hormone replacement therapy isemployed.

The present invention is explained in greater detail in the followingnon-limiting Examples.

Example 1 Isolation of Rat Ovaries

As schematically illustrated in FIG. 1, Panel A, postnatal day 21Fischer 344 rats were injected with 1.5 mg/0.2 ml of 17β-estradiol (E2)dissolved in sesame oil, subcutaneously for three consecutive days. Therats were euthanized 24 h after the last injection, ovaries were excisedand endocrine cells were isolated as described in Example 2:

Example 2 Cell Isolation and Purification

The endocrine cells were isolated from ovaries of E2-primed immaturerats according to Li and Hearn (J. Biochem. Biophys. Methods 45, 169-181(2000). Ovaries collected in ice cold medium 199 (M199) containing HEPES(25 mM), 1 mg/ml bovine serum albumin (BSA), L-glutamine (2 mM),penicillin (10,000 IU/ml), streptomycin (10,000 μg/ml), and amphotericinB (25 μg/ml). After cleaning the extraneous tissues, the ovaries werewashed twice with ice cold M199 and then punctured gently with 27Gsyringe needles in order to release the loosely packed granulosa fromthe follicles; cells thus collected were kept on ice. The remainingovaries were chopped into fine pieces of ˜0.25 mm² and the cellsreleased during this process were collected and kept on ice separately.The pieces of ovaries were then incubated with collagenase (2 mg/ml) andDNase (10 μg/ml) in M199 for 90 min with occasional mixing. Theenzyme-digested pieces were dispersed using a Pasteur pipette to obtaina single cell suspension and collected and stored on ice as a separatefraction. Cells from different fractions collected above were purifiedas per Magoffin and Erickson (Endocrinology 122, 2345-2347 (1988)).Briefly, the cells were loaded on top of a discontinuous percollgradient (44% in the bottom, d=1.055 percoll (specific gravity adjustedto 1.055) in the middle and 20% on the top) and centrifuged at 400×g for20 minutes at 4° C. Cells from the first interphase (between 20% andd=1.055 layers) were recovered as granulosa cells and those from thesecond interphase (between d=1.055 and 44% layers) were collected astheca cells (See FIG. 1, Panel D). The viability of the cells waschecked using the trypan blue method and was in the 85-95% range. Thepurity of each cell type was assessed by flow cytometric analysis usingcell-specific markers.

Example 3 Cells Analyzed Using Flow Cytometry

A fraction of the cells (5×10⁶ cells/cell type) purified using thediscontinuous percoll gradient was fixed in 3.7% formaldehyde for 15minutes.

To verify the purity of the cell types isolated from the rat ovaries,the cells were stained with cell-specific markers and quantified by flowcytometry. Cells from different interphases (See FIG. 1, Panel D) wereincubated with primary antibodies. Antibody for CYP19 (mouse anti-CYP19;Abbiotech; cat. 250549) and FITC-conjugated secondary antibody were usedto detect the granulosa cells. Antibody for CYP17A1 (goat anti-CYP17A1;Santa Cruz Biotechnology; cat. sc-46085) and PerCP Cy5.5-conjugateddonkey anti-goat IgG secondary antibody were used to detect the thecacells. Cells were incubated with the appropriate primary antibody for 1h. Unbound antibodies were then washed off and the cells were incubatedwith the appropriate secondary antibody for 1 h. After washing off theunbound secondary antibodies, cells were analyzed using flow cytometry.The flow cytometric analysis revealed that 74.15% of the cells recoveredfrom the first interphase in the percoll gradient stained positive forCYP19 (FIG. 1, Panel B) and 69.91% of the cells obtained from the secondinterphase stained for CYP17A1 (FIG. 1, Panel C). Cells incubated withonly secondary antibodies were used as control.

Example 4 Culture of Granulosa and Theca Cells

Purified granulosa and theca cells were separately incubated at 37° C.under an atmosphere of 5% CO₂ in humidified air in T175 flasks (Corning,Corning Inc., NY, USA) cultured for 24 h in McCoy's 5A medium supplementwith L-glutamine (2 mM), penicillin (10,000 IU/ml), streptomycin (10,000μg/ml), amphotericin B (25 μg/ml) and 10% FBS. The medium for granulosacells was replaced with granulosa growth medium (McCoy's 5A withL-glutamine (2 mM), BSA (1 mg/ml), penicillin (10,000 IU/ml),streptomycin (10,000 μg/ml), and amphotericin B (25 μg/ml), 200 ng/mloFSH, 100 nM E2 and 10 nM IGF-I) and cultured for an additional 72 h.Similarly, the theca cells were grown for another 72 h in theca growthmedium (McCoy's 5A medium supplemented with L-glutamine (2 mM), BSA (1mg/ml), penicillin (10,000 IU/ml), streptomycin (10,000 μg/ml),amphotericin B (25 μg/ml), 100 ng/ml oLH; 10 nM IGF-I).

Example 5 Immuno-Fluorescence Staining

Each cell type was cultured on chamber slides in respective growthmedium and screened for the expression of essential cellular componentsfor steroidogenesis. After fixing the cells in 3.7% formaldehyde for 15minutes, cells were washed with PBS and blocked with PBS with BSA (1%).The monolayer was then incubated with primary antibodies overnight at 4°C. Granulosa cells were incubated with rabbit anti-FSHR (Santa CruzBiotechnology; cat. no. sc-13935) and mouse anti-CYP19 (Abbiotech; cat.no. 250549). Similarly theca cells were incubated with rabbit anti-LHR(Santa Cruz Biotechnology; cat. no. sc-25828) and goat anti-CYP17A1(Santa Cruz Biotechnology; cat. no. sc-46085). After overnightincubation with primary antibodies, the slides were washed with PBS andincubated with secondary antibodies for 2 h at 4° C. The unboundsecondary antibodies were washed away and the nucleus was counterstainedwith DAPI and cover slips were mounted. The images were acquired using afluorescence microscope and composite images were made with the help ofImage-Pro plus software version 6.3.1.542.

While theca cells stained positive for LH-receptor (LHR) and CYP17A1(FIG. 2), granulosa cells showed positive for FSH-receptor (FSHR) andCYP19 (FIG. 2).

Example 6 Granulosa Cells and Theca Cells Encapsulated Separately

Cultured cells were encapsulated separately by extrusion through amulti-nozzle extruder in 1 to 3% (w/v) ultrapure low viscosityhigh-mannuronic (LVM) alginate solution into calcium chloride solutionfor 5 to 15 minutes (for cross-linking) to produce microcapsules ofapproximately 300 to 600 micron diameter. All the encapsulation andwashing steps are carried out at room temperature. Granulosacell-containing microcapsules and theca cell-containing microcapsuleswere then combined together with one another in equal parts, co-culturedtogether in separate chambers of culture inserts in 24-well plates inMcCoy's 5A medium supplemented with penicillin/streptomycin (100 IU/ml &100 μg/ml, respectively), amphotericin B (0.25 μg/ml) and fetal bovineserum (10%) at 37° C. and 5% CO₂. The viability and 17β-estradiolproduction as discussed below was evaluated periodically for 30 days.

The microcapsules received 50 ng/ml follicle-stimulating hormone (FSH)and 50 ng/ml luteinizing hormone (LH) in long-term cultures. LHtreatment increased the expression of CYP17A1 (17, 20 lyase) in thecacells and FSH treatment increased the expression of CYP19 (aromatase) ingranulosa cells in vitro (FIG. 2), which improves the steroidogenicpotency of these cells. Encapsulation distributed cells evenly in thealginate microcapsules (FIG. 3). It was noted that optimum cell densityis an important factor for configuration and structure of themicrocapsule, which was approximately 1,000 to 10,000 cells permicrocapsule.

Encapsulated cells had sustained viability during the long-term cultureup to day 30 (See Example 10 and FIG. 4). The number of non-viable cellsincreased in the course of long-term culture.

Note that granulosa cell-containing microcapsules co-cultured with thecacell-containing microcapsules produced significantly higher levels of E2than either cultured individually (FIG. 5, Panel A).

In addition, co-culture of granulosa cell-containing microcapsules withtheca cell-containing microcapsules secreted increased levels of E2 inresponse to FSH and LH in the long-term culture in vitro (FIG. 5, PanelB and Panel C).

These data show that ovarian endocrine cells encapsulated in alginatehydrogel microcapsules showed both long-term survival and bioactivity invitro. With the encapsulation technique we were able to demonstrate thatthe endocrine unit of ovaries could be recapitulated ex vivo.

In additional experiments, portions of microcapsules were cultured inthe presence of FSH (100 ng/ml) and LH (100 ng/ml) for about 30 days andthe culture media were collected every alternate day to test thesecretion of sex steroids. The levels of 17β-estradiol and progesteronein the culture media were quantified using ELISA kits. 17β-estradiol inculture media was measured with an ELISA kit from Enzo Life Sciences(cat. No. ADI-901-008). The progesterone levels in cell culture mediawere measured using the ELISA kit from Enzo Life Sciences (cat. no.ADI-901-011). The levels of 17β-estradiol and progesterone werequantified according to the manufacturer's instructions and correctedfor their dilutions.

When granulosa cell-containing microcapsules or theca cell-containingmicrocapsules were incubated separately, there were no significantincreases in the production of 17β-estradiol. In the same experiments,the progesterone levels reached 1.3 and 0.8 ng/ml at days 4 and 6,respectively (See FIG. 5, Panel D and Panel E).

When granulosa cell-containing microcapsules and theca cell-containingmicrocapsules were co-cultured, the 17β-estradiol level reached ˜20pg/ml at day 18 and the progesterone level peaked at ˜1.5 ng/ml at day26 (See FIG. 5, Panel D and Panel E).

Example 7 Granulosa Cells and Theca Cells Encapsulated Together

This example is carried out in like manner as Example 6 above, exceptthat the granulosa and theca cells are mixed together in essentiallyequal amounts prior to extrusion, so that the two are encapsulatedtogether.

A portion of microcapsules were cultured in the presence of FSH (100ng/ml) and LH (100 ng/ml) for about 30 days and the culture media werecollected every alternate day to test the secretion of sex steroids. Thelevels of 17β-estradiol and progesterone in the culture media werequantified using ELISA kits. 17β-Estradiol in culture media was measuredwith an ELISA kit from Enzo Life Sciences (cat. No. ADI-901-008). Theprogesterone levels in cell culture media were measured using the ELISAkit from Enzo Life Sciences (cat. no. ADI-901-011). The levels of17β-estradiol and progesterone were quantified according to themanufacturer's instructions and corrected for their dilutions.Encapsulated cells responded to the gonadotropins from day 2 onward. The17β-estradiol levels were approximately 5-fold higher by day 25, whencompared to basal levels, and the progesterone levels were approximately2 fold higher when compared to basal levels; see FIG. 5, Panel D andPanel E.

Example 8 Porcine Bone Marrow Stromal Cells Encapsulated Together inLayers

Two layer microcapsules (schematically illustrated in FIG. 6) wereproduced in accordance with the technique described in O. Khanna et al.,J. Biomed. Mater. Res. Part A 95A: 632-640 (2010). Briefly, porcine bonemarrow stromal cells (pBMSC) were cultured in DMEM supplemented withpenicillin/streptomycin (100 IU/ml & 100 μg/ml, respectively),amphotericin B (0.25 μg/ml), fetal bovine serum (10%) at 37° C. and 5%CO₂ and tagged with vital fluorescent probe CellTracker green andCellTracker orange (invitrogen). pBMSC probed with CellTracker greenwere encapsulated in 1-2% low viscosity high-mannuronic (LVM) alginateby extrusion through a multi-nozzle extruder into a calcium chloridesolution. The microcapsules were then suspended with a 0.05 to 0.2%poly-L-ornithine solution for about 5 to 30 minutes at 4° C. to createthe permselective membrane layer. The coated microcapsules were thencoated with a second layer of alginate, which was 0.5 to 2% (w/v) lowviscosity high-glucoronic alginate (LVG) containing CellTrackerorange-probed pBMSC. About 1,000 to 10,000 cells are included in eachlayer of the capsule.

Example 9 Granulosa Cells and Theca Cells Encapsulated in a Two LayerMicrocapsule

Granulosa cells were encapsulated in 1.5% (w/v) LVM and coated withpoly-L-ornithine (PLO) (0.1% w/v) for 20 minutes. The PLO-coatedmicrocapsules were then mixed with theca cells suspended in 1.5% (w/v)LVM and encapsulated again using the micro-fluidic device (FIG. 1, PanelA) in order to obtain multi-layered microcapsules, which resemble thestructural architecture of native follicles as depicted in FIG. 6(referred to as multi-layered microcapsules).

A portion of microcapsules were cultured in the presence of FSH (100ng/ml) and LH (100 ng/ml) for about 30 days and the culture media werecollected every alternate day to test the secretion of sex steroids. Thelevels of 17β-estradiol and progesterone in the culture media werequantified using ELISA kits. 17β-estradiol in culture media was measuredwith an ELISA kit from Enzo Life Sciences (cat. No. ADI-901-008). Theprogesterone levels in cell culture media were measured using the ELISAkit from Enzo Life Sciences (cat. no. ADI-901-011). The levels of17β-estradiol and progesterone were quantified according to themanufacturer's instructions and corrected for their dilutions. There wasa ten-fold increase in the 17β-estradiol by day 25 and progesteronelevels were approximately 2 fold higher when compared to basal levels,see FIG. 5, Panel D and Panel E.

To demonstrate the differential compartmentalization of different celltypes in the multi-layered microcapsules, the granulosa cells werepre-stained with Cell Tracker green (Invitrogen, cat. No. C2925) and thetheca cells were pre-stained with Cell-tracker Orange (Invitrogen, cat.No. C2927), prior to the synthesis of the multi-layered microcapsules.The multi-layered microcapsules were imaged using a confocal microscope(Zeiss LSM510).

Example 10 Viability of Encapsulated Ovarian Endocrine Cells

The viability of the encapsulate cells were assessed using live deadanalysis. A portion of microcapsules from Examples 6, 7, 8, and 9 werecultured in the presence of FSH (100 ng/ml) and LH (100 ng/ml) for about30 days and the culture media were collected approximately every thirdday to test the viability of the encapsulated cells. At the designatedtimes, encapsulated cells were transferred to a 24-well plate andincubated with 25 μM CFDA SE (carboxyfluorescein diacetate, succinimidylester) (Invitrogen, cat. no. V12883) in serum-free medium for 15 minutesat 37° C. under an atmosphere of 5% CO₂ in humidified air. Then the CFDAcontaining medium was replace with medium containing 10% FBS andincubated again under the above-mentioned conditions for an additional30 min. The serum-containing medium was then replace with 50 μg/ml ofpropidium iodide (PI) (Invitrogen, cat. no. V12883) and incubated atroom temperature for 2 min and the microcapsules were washed to removeexcess PI. The microcapsules were then observed under an invertedfluorescence microscope and imaged. The number of live and dead cellswas analyzed from the acquired composite image using Image-Pro plussoftware version 6.3.1.542.

Note: live cells cleave the ester group of membrane permeablenon-fluorescent CFDA and convert it into non-permeable-green fluorescentFDA, which gets trapped inside viable cells. On the other hand, deadcells have a compromised membrane whereby propidium penetrates into thenucleus and stains the DNA red. The periodical live/dead analysisrevealed the encapsulated ovarian endocrine cells had a sustainedviability throughout the period of long-term culture (See FIG. 4).

Example 11 In Vivo Functions of Tissue-Engineered Ovarian Endocrine Unit

Ovariectomy and implantation of multi-layered microcapsules: Six monthold Fisher 344 rats were ovariectomized bilaterally under anaesthesiaand their blood levels of E₂ and P₄ were followed until they reach thebasal levels. Once the basal levels were reached the multi-layeredmicrocapsules, from Example 9, were implanted in a pouch made from thegreat omentum mesentery of the ovx rats. Approximately 000 microcapsuleslodging 0.5×10⁶ cells of each cell type were implanted in each of theexperimental rats (n=5). The control rats (n=5) received an equivalentnumber of blank alginate microcapsules in the omental pouch. The levelsof 17β-estradiol and progesterone in the blood plasma were quantifiedusing ELISA kits. 17β-Estradiol in plasma was measured using ELISA kitsfrom Enzo Life Sciences (cat. no. ADI-901-174). The progesterone levelsin blood plasma were measured using the ELISA kit (Enzo, Life sciences,cat. no. ADI-901-011). The levels of 17β-estradiol and progesterone werequantified according to the manufacturer's instructions and correctedfor their dilutions. Compared to the ovx control rats, the plasma levelsof 17β-estradiol in the tissue-construct transplanted rats weresignificantly higher in all the time points measured (FIG. 7A).Similarly the progesterone levels were also significantly higher thanthat of the control rats (FIG. 7B).

Student's t-tests were performed for the in vivo study to comparebetween the means of hormone levels of microcapsule-implanted rats andthat of ovx rats.

Example 12 Long-Term Co-Culture in a 2D System

Following isolation, granulosa and theca cells were either culturedseparately or co-cultured in a 2D system in the presence of thegonadotrophins, LH and FSH (See FIGS. 8A and 8B). In this example, thelevels of progesterone increased with time in the culture media (from0.2 ng/ml on day 2 to ˜2 ng/ml on day 30) while the level of17β-estradiol decreased from 28 pg/ml on day 2 to ˜5 pg/ml on day 30.These data indicate abnormal cell responses in which the granulosa cellslose their estrogenic potential to acquire progesteronic potentialthereby enhancing the observed levels of progesterone in the media, incontrast to the normal physiological responses of these cells in thenative ovaries.

Example 13 Statistical Analysis

Statistical analyses were performed using SPSS software (version10.0.1). Results are presented as the mean±S.E.M unless statedotherwise. For the in vitro study, comparisons between the means ofhormone levels of three different schemes and the control groups wereperformed using analysis of variance (ANOVA) followed by post-hoctesting using, when appropriate, Bonferroni correction. Differences wereconsidered to be statistically significant when P<0.05.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A pharmaceutical composition comprisingmicrocapsules, said microcapsules containing both live mammalian ovariangranulosa cells and live mammalian ovarian theca cells; wherein saidmicrocapsules comprise a core and an auxiliary layer surrounding saidcore, said core containing said granulosa cells and said auxiliary layercontaining said theca cells; and wherein said microcapsules are free ofoocytes.
 2. The composition of claim 1, said microcapsules furthercomprising a first semipermeable layer between said core and saidauxiliary layer.
 3. The composition of claim 2, said microcapsulesfurther comprising a second semipermeable layer surrounding saidauxiliary layer.
 4. The composition of claim 3, said microcapsulesfurther comprising an external polysaccharide layer surrounding saidsecond semipermeable layer.
 5. The composition of claim 4, wherein saidsemipermeable layers are formed of a polycation.
 6. The composition ofclaim 5, wherein said polycation is a polyamine.
 7. The composition ofclaim 1, wherein said microcapsules comprise a hydrogel.
 8. Thecomposition of claim 7, wherein said hydrogel comprises a polysaccharidehydrogel.
 9. The composition of claim 1, wherein said microcapsules arefrom 10 microns in diameter, up to 5000 microns in diameter.
 10. Thecomposition of claim 1, wherein said granulosa cells are included insaid microcapsules in an amount of from 1,000 cells per microcapsule upto 1×10⁹ cells per microcapsule; and said theca cells are included insaid microcapsules an amount of from 1,000 cells per microcapsule up to1×10⁹ cells per microcapsule.
 11. The composition of claim 6, whereinsaid microcapsules comprise a hydrogel.
 12. The composition of claim 11,wherein said hydrogel comprises a polysaccharide hydrogel.
 13. Thecomposition of claim 6, wherein said microcapsules are from 10 micronsin diameter, up to 5000 microns in diameter.
 14. The composition ofclaim 6, wherein: said granulosa cells are included in saidmicrocapsules in an amount of from 1,000 cells per microcapsule up to1×10⁹ cells per microcapsule; and said theca cells are included in saidmicrocapsules an amount of from 1,000 cells per microcapsule up to 1×10⁹cells per microcapsule.
 15. The composition of claim 1, wherein saidmicrocapsules produce estrogen.
 16. The composition of claim 15, whereinsaid microcapsules further produce progesterone.
 17. The composition ofclaim 6, wherein said microcapsules produce estrogen.
 18. Thecomposition of claim 17, wherein said microcapsules further produceprogesterone.